G protein-coupled receptors (GPCRs) are major drug targets for a wide spectrum of diseases including neurological diseases such as migraine, anxiety, psychosis, and schizophrenia Novel robust assay technologies are continuously demanded for GPCR targets to increase odds for leads generation in a large-scale compound screening. Activation of GPCRs results in changes in intracellular calcium and cAMP About 70 percent of nonchemosensory GPCRs mediate their signals through up- or down-regulation of intracellular Camp via Gs and Gi proteins respectively We propose to develop a high-throughput technology for real time assay of Gs protein-coupled receptors (Gs-GPCRs) In this application, cyclic nucleotide-gated (CNG) channel variants are specifically used as a physiological sensor for cAMP, coupling a change in intracellular cAMP concentration to changes in cytosolic calcium concentration and membrane potential Our results demonstrate that the cAMP change induced by activation of adenylyl cyclase and GPCRs can be sensitively monitored in the living mammalian cells expressing the CNG channel variants by using calcium and voltage indicators Stable cells with the sensitive CNG channel mutants have been developed to enable the assay in 96-well microplates This convenient assay allows for the kinetic detection of transient cAMP signals with a high signal-tonoise ratio CNG channels may also provide a means to detect local cAMP concentrations in proximity to adenylylcyclase, allowing for a sensitive detection of GPCR activity Towards next stage of commercialization, we propose to further improve and adapt this robust GPCR assay to standard high-throughput screening platforms including FLIPR.